Method of sorting polynucleotides

ABSTRACT

The invention provides a method and materials for sorting polynucleotides with oligonucleotide tags. Oligonucleotide tags of the invention are capable of hybridizing to complementary oligomeric compounds consisting of subunits having enhanced binding strength and specificity as compared to natural oligonucleotides. Such complementary oligomeric compounds are referred to herein as &#34;tag complements.&#34; Subunits of tag complements may consist of monomers of non-natural nucleotide analogs, referred to herein as &#34;antisense monomers&#34; or they may comprise oligomers having lengths in the range of 3 to 6 nucleotides or analogs thereof, including antisense monomers, the oligomers being selected from a minimally cross-hybridizing set. In such a set, a duplex made up of an oligomer of the set and the complement of any other oligomer of the set contains at least two mismatches. Preferred antisense monomers include peptide nucleic acid monomers and nucleoside phosphoramidates having a 3&#39;--NHP(O)(O--)O--5&#39; linkage with its adjacent nucleoside. 
     An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, or the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

This is a continuation of U.S. patent application Ser. No. 08/359,295filed Dec. 19, 1994, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/322,348 filed Oct. 13, 1994, now abandoned,which applications are incorporated by reference.

FIELD OF THE INVENTION

The invention relates generally to methods for sequencingpolynucleotides, and more particularly, to a method of sorting andsequencing many polynucleotides simultaneously.

BACKGROUND

Presently there are two basic approaches to DNA sequence determination:the chain termination method, e.g. Sanger et al, Proc. Natl. Acad. Sci.,74: 5463-5467 (1977); and the chemical degradation method, e.g. Maxam etal, Proc. Natl. Acad. Sci., 74: 560-564 (1977). The chain terminationmethod has been improved in many ways since its invention, and serves asthe basis for all currently available automated DNA sequencing machines,e.g. Sanger et al, J. Mol. Biol., 143: 161-178 (1980); Schreier et al,J. Mol. Biol., 129: 169-172 (1979); Smith et al, Nature, 321: 674-679(1987); Prober et al, Science, 238: 336-341 (1987); Hunkapiller et al,Science, 254: 59-67 (1991); Bevan et al, PCR Methods and Applications,1: 222-228 (1992). Moreover, further improvements are easily envisionedthat should greatly enhance the throughput and efficiency of theapproach, e.g. Huang et al, Anal. Chem., 64: 2149-2154 (1992)(capillaryarrays); Best et al, Anal. Chem., 66: 4063-4067 (1994)(non-cross-linkedpolymeric separation media for capillaries); better dye sets; and thelike.

Nonetheless, even with such reasonably envisioned improvements, theseapproaches still have several inherent technical problems that make themboth expensive and time consuming, particularly when applied tolarge-scale sequencing projects. Such problems include i) the gelelectrophoretic separation step which is labor intensive, is difficultto automate, and which introduces an extra degree of variability in theanalysis of data, e.g. band broadening due to temperature effects,compressions due to secondary structure in the DNA sequencing fragments,inhomogeneities in the separation gel, and the like; ii) nucleic acidpolymerases whose properties, such as processivity, fidelity, rate ofpolymerization, rate of incorporation of chain terminators, and thelike, are often sequence dependent; iii) detection and analysis of DNAsequencing fragments which are typically present in fmol quantities inspacially overlapping bands in a gel; iv) lower signals because thelabelling moiety is distributed over the many hundred spaciallyseparated bands rather than being concentrated in a single homogeneousphase, v) in the case of single-lane fluorescence detection, theavailability of dyes with suitable emission and absorption properties,quantum yield, and spectral resolvability; and vi) the need for aseparately prepared sequencing template for each sequencing reaction toidentify a maximum of about 400-600 bases, e.g. Trainor, Anal. Biochem.,62: 418-426 (1990); Connell et al, Biotechniques, 5: 342-348 (1987);Karger et al, Nucleic Acids Research, 19: 4955-4962 (1991); Fung et alU.S. Pat. No. 4,855,225; Nishikawa et al, Electrophoresis, 12: 623-631(1991); and Hunkapiller et al (cited above).

The need to prepare separate sequencing templates is especially onerousin large-scale sequencing projects, e.g. Hunkapiller et al (citedabove)(94.4 kilobase target--2399 templates); and Alderton et at, Anal.Biochem., 201: 166-169 (1992)(230 kilobase target--13,000 templates).

Attempts to automate template preparation have proved difficult,especially when coupled with current sequencing methodolgies, e.g.Church et al, Science, 240: 185-188 (1988); Beck et al, Anal. Biochem.212: 498-505 (1993); Wilson et al, Biotechniques, 6: 776-787 (1988); andthe like.

In view of the above, a major advance in sequencing technology wouldtake place if there were means available for overcoming thetemplate-preparation bottleneck. In particular, the ability to preparemany thousands of templates simulaneously without individual templateselection and handling would lead to significant increases in sequencingthroughput and significant lowering of sequencing costs.

SUMMARY OF THE INVENTION

An object of my invention is to provide a method for tagging and sortingmany thousands of fragments of a target polynucleotide for simultaneousanalysis and/or sequencing.

Another object of my invention is to provide a method, kits, andapparatus for analyzing and/or sequencing many thousands of differentpolynucleotides simultaneously.

A further object of my invention is to provide a method for greatlyreducing the number of separate template preparation steps required inlarge scale sequencing projects.

Still another object of my invention is to provide a method for applyingsingle-base sequencing methodologies to many different targetpolynucleotides simultaneously.

Another object of my invention is to provide a rapid and reliable methodfor sequencing target polynucleotides having a length in the range of afew hundred basepairs to several tens of thousands of basepairs.

My invention achieves these and other objects by providing a method andmaterials for sorting polynucleotides with oligonucleotide tags.Oligonucleotide tags of the invention are capable of hybridizing tocomplementary oligomeric compounds consisting of subunits havingenhanced binding strength and specificity as compared to naturaloligonucleotides. Such complementary oligomeric compounds are referredto herein as "tag complements." Subunits of tag complements may consistof monomers of non-natural nucleotide analogs, referred to herein as"antisense monomers" or they may comprise oligomers having lengths inthe range of 3 to 6 nucleotides or analogs thereof, including antisensemonomers, the oligomers being selected from a minimallycross-hybridizing set. In such a set, a duplex made up of an oligomer ofthe set and the complement of any other oligomer of the set contains atleast two mismatches. In other words, an oligomer of a minimallycross-hybridizing set at best forms a duplex having at least twomismatches with the complement of any other oligomer of the same set.The number of oligonucleotide tags available in a particular embodimentdepends on the number of subunits per tag and on the length of thesubunit, when the subunit is an oligomer from a minimallycross-hybridizing set. In the latter case, the number is generally muchless than the number of all possible sequences the length of the tag,which for a tag n nucleotides long would be 4^(n). Preferred antisensemonomers include peptide nucleic acid monomers and nucleosidephosphoramidates having a 3'--NHP(O)(O--)O--5' linkage with its adjacentnucleoside. The latter compounds are referred to herein as N3'→P5'phosphoramidates.

In one aspect of my invention, tag complements attached to a solid phasesupport are used to sort polynucleotides from a mixture ofpolynucleotides each containing a tag. In this embodiment, tagcomplements are synthesized on the surface of a solid phase support,such as a microscopic bead or a specific location in an array ofsynthesis locations on a single support, such that populations ofidentical sequences are produced in specific regions. That is, thesurface of each support, in the case of a bead, or of each region, inthe case of an array, is derivatized by only one type of tag complementwhich has a particular sequence. The population of such beads or regionscontains a repertoire of tag complements with distinct sequences, thesize of the repertoire depending on the number of subunits peroligonucleotide tag and the length of the subunits employed, whereoligomeric subunits are used. Similarly, the polynucleotides to besorted each comprises an oligonucleotide tag in the repertoire, suchthat identical polynucleotides have the same tag and differentpolynucleotides have different tags. Thus, when the populations ofsupports and polynucleotides are mixed under conditions which permitspecific hybridization of the oligonucleotide tags with their respectivecomplements, subpopulations of identical polynucleotides are sorted ontoparticular beads or regions. The subpopulations of polynucleotides canthen be manipulated on the solid phase support by micro-biochemicaltechniques.

An important aspect of my invention is the use of the oligonucleotidetags to sort polynucleotides for parallel sequence determination.Preferably, this aspect of the invention comprises the following steps:(a) generating from a target polynucleotide a plurality of fragmentsthat covers the target polynucleotide; (b) attaching an oligonucleotidetag from a repertoire of tags to each fragment of the plurality (i) suchthat substantially all the same fragments have the same oligonucleotidetag attached and (ii) such that each oligonucleotide tag from therepertoire comprises a plurality of subunits and each subunit of theplurality consists of a complementary nucleotide of an antisense monomeror an oligonucleotide having a length from three to six nucleotides, theoligonucleotides being selected from a minimally cross-hybridizing set;(c) sorting the fragments by specifically hybridizing theoligonucleotide tags with their respective tag complements; (d)determining the nucleotide sequence of a portion of each of thefragments of the plurality; and (e) determining the nucleotide sequenceof the target polynucleotide by collating the sequences of thefragments.

Another important feature of my invention is a method of identifying, orfingerprinting, a population of mRNA molecules. Preferably, such amethod comprises the following steps: (a) forming a population of cDNAmolecules from the population of mRNA molecules, the cDNA moleculesbeing complementary to the mRNA molecules and each cDNA molecule havingan oligonucleotide tag attached, (i) such that substantially all of thesame cDNA molecules have the same oligonucleotide tag attached and (ii)such that each oligonucleotide tag from the repertoire comprises aplurality of subunits and each subunit of the plurality consists of acomplementary nucleotide of an antisense monomer or an oligonucleotidehaving a length from three to six nucleotides, the oligonucleotidesbeing selected from a minimally cross-hybridizing set; (b) sorting thecDNA molecules by specifically hybridizing the oligonucleotide tags withtheir respective tag complements; (c) determining the nucleotidesequence of a portion of each of the sorted cDNA molecules; and (d)identifying the population of mRNA molecules by the frequencydistribution of the portions of sequences of the cDNA molecules.

When used in combination with solid phase supports, such as microscopicbeads, my invention provides a readily automated system for manipulatingand sorting polynucleotides, particularly useful in large-scale paralleloperations, such as large-scale DNA sequencing, mRNA fingerprinting, andthe like, where many target polynucleotides or many segments of a singletarget polynucleotide are sequenced and/or analyzed simultaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1c illustrates structures of labeled probes employed in apreferred method of "single base" sequencing which may be used with theinvention.

FIG. 2 illustrates the relative positions of the nuclease recognitionsite, ligation site, and cleavage site in a ligated complex formedbetween a target polynucleotide and a probe (SEQ ID NO: 22)used in apreferred "single base" sequencing method.

FIG. 3 is a flow chart illustrating a general algorithm for generatingminimally cross-hybridizing sets.

FIG. 4 illustrates a scheme for synthesizing oligonucleotide N3'→P5'phosphoramidates.

FIG. 5 diagrammatically illustrates an apparatus for carrying outparallel operations, such as polynucleotide sequencing, in accordancewith the invention.

DEFINITIONS

"Complement" or "tag complement" as used herein in reference tooligonucleotide tags refers to an oligonucleotide to which aoligonucleotide tag specifically hybridizes to form a perfectly matchedduplex or triplex. In embodiments where specific hybridization resultsin a triplex, the oligonucleotide tag may be selected to be eitherdouble stranded or single stranded. Thus, where triplexes are formed,the term "complement" is meant to encompass either a double strandedcomplement of a single stranded oligonucleotide tag or a single strandedcomplement of a double stranded oligonucleotide tag.

The term "oligonucleotide" as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, a-anomeric forms thereof, peptidenucleic acids (PNAs), and the like, capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like.Usually monomers are linked by phosphodiester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g. 3-4, to several tens of monomeric units. Whenever anoligonucleotide is represented by a sequence of letters, such as"ATGCCTG," it will be understood that the nucleotides are in 5'→3' orderfrom left to right and that "A" denotes deoxyadenosine, "C" denotesdeoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine,unless otherwise noted. Analogs of phosphodiester linkages includephosphorothioate, phosphorodithioate, phosphoranilidate,phosphoramidate, and the like. It is clear to those skilled in the artwhen oligonucleotides having natural or non-natural nucleotides may beemployed, e.g. where processing by enzymes is called for, usuallyoligonucleotides consisting of natural nucleotides are required."Perfectly matched" in reference to a duplex means that the poly- oroligonucleotide strands making up the duplex form a double strandedstructure with one other such that every nucleotide in each strandundergoes Watson-Crick basepairing with a nucleotide in the otherstrand. The term also comprehends the pairing of nucleoside analogs,such as deoxyinosine, nucleosides with 2-aminopurine bases, and thelike, that may be employed. In reference to a triplex, the term meansthat the triplex consists of a perfectly matched duplex and a thirdstrand in which every nucleotide undergoes Hoogsteen or reverseHoogsteen association with a basepair of the perfectly matched duplex.Conversely, a "mismatch" in a duplex between a tag and anoligonucleotide means that a pair or triplet of nucleotides in theduplex or triplex fails to undergo Watson-Crick and/or Hoogsteen and/orreverse Hoogsteen bonding.

As used herein, "nucleoside" includes the natural nucleosides, including2'-deoxy and 2'-hydroxyl forms, e.g. as described in Komberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). "Analogs" inreference to nucleosides includes synthetic nucleosides having modifiedbase moieties and/or modified sugar moieties, e.g. described by Scheit,Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman,Chemical Reviews, 90: 543-584 (1990), or the like, with the only provisothat they are capable of specific hybridization. Such analogs includesynthetic nucleosides designed to enhance binding properties, reducedegeneracy, increase specificity, and the like.

"Stable" in reference to the formation of a covalent linkage and/ornon-covalent complex between binding moieties means that meltingtemperature of the oligonucleotide clamp incorporating the given pair(s)of binding moieties and its target polynucleotide is increased by atleast twenty-five percent over the melting temperature ofoligonucleotide moieties of the clamp alone, wherein melting temperatureis measured by standard techniques, e.g. half maximum of 260 nmabsorbance v. temperature as described more ally below. Preferably,stable means that melting temperature of the oligonucleotide clampincorporating the given pair(s) of binding moieties and its targetpolynucleotide is increased by at least fifty percent over the meltingtemperature of oligonucleotide moieties of the clamp alone.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of labeling and sorting molecules,particularly polynucleotides, by the use of oligonucleotide tags. In oneaspect, the oligonucleotide tags of the invention comprise a pluralityof "words" or subunits selected from minimally cross-hybridizing sets ofsubunits. Subunits of such sets cannot form a duplex or triplex with thecomplement of another subunit of the same set with less than twomismatched nucleotides. Thus, the sequences of any two oligonucleotidetags of a repertoire that form duplexes will never be "closer" thandiffering by two nucleotides. In particular embodiments, sequences ofany two oligonucleotide tags of a repertoire can be even "further"apart, e.g. by designing a minimally cross-hybridizing set such thatsubunits cannot form a duplex with the complement of another subunit ofthe same set with less than three mismatched nucleotides, and so on.Usually, oligonucleotide tags of the invention and their complements areoligomers of the natural nucleotides so that they may be convenientlyprocessed by enzymes, such as ligases, polymerase, terminaltransferases, and the like.

In another aspect of the invention, tag complements consist of monomersreferred to herein as "antisense monomers." This term is meant toencompass a range of compounds typically developed for antisensetherapeutics that have enhance binding strength and enhance specificityfor polynucleotide targets. As mentioned above under the definition of"oligonucleotide," the compounds may include a variety of differentmodifications of the natural nucleotides, e.g. modification of basemoieties, sugar moieties, and/or monomer-to-monomer linkages. Suchcompounds also include oligonucleotide loops, oligonucleotide "clamps,"and like structures, described more fully below, that promote enhancedbinding and specificity.

By the use of such compounds, the invention finds particular utility inlabeling and sorting polynucleotides for parallel operations, such assequencing, fingerprinting or other types of analysis.

Constructing Oligonucleotide Tags from Minimally Cross-Hybridizing Setsof Subunits

The nucleotide sequences of the subunits for any minimallycross-hybridizing set are conveniently enumerated by simple computerprograms following the general algorithm illustrated in FIG. 3, and asexemplified by program minhx whose source code is listed in Appendix I.Minhx computes all minimally cross-hybridizing sets having subunitscomposed of three kinds of nucleotides and having length of four.

The algorithm of FIG. 3 is implemented by first defining thecharacteristic of the subunits of the minimally cross-hybridizing set,i.e. length, number of base differences between members, andcomposition, e.g. do they consist of two, three, or four kinds of bases.A table M_(n), n=1, is generated (100) that consists of all possiblesequences of a given length and composition. An initial subunit S₁ isselected and compared (120) with successive subunits S_(i) for i=n+1 tothe end of the table. Whenever a successive subunit has the requirednumber of mismatches to be a member of the minimally cross-hybridizingset, it is saved in a new table M_(n+1) (125), that also containssubunits previously selected in prior passes through step 120. Forexample, in the first set of comparisons, M₂ will contain S₁ ; in thesecond set of comparisons, M₃ will contain S₁ and S₂ ; in the third setof comparisons, M₄ will contain S₁, S₂, and S₃ ; and so on. Similarly,comparisons in table M_(j) will be between S_(j) and all successivesubunits in M_(j). Note that each successive table M_(n+1) is smallerthan its predecessors as subunits are eliminated in successive passesthrough step 130. After every subunit of table M_(n) has been compared(140) the old table is replaced by the new table M_(n+1), and the nextround of comparisons are begun. The process stops (160) when a tableM_(n) is reached that contains no successive subunits to compare to theselected subunit S_(i), i.e.

M_(n) =M_(n+1).

As mentioned above, preferred minimally cross-hybridizing sets comprisesubunits that make approximately equivalent contributions to duplexstability as every other subunit in the set. Guidance for selecting suchsets is provided by published techniques for selecting optimal PCRprimers and calculating duplex stabilities, e.g. Rychlik et al, NucleicAcids Research, 17: 8543-8551 (1989) and 18: 6409-6412 (1990); Breslaueret al, Proc. Natl. Acad. Sci., 83: 3746-3750 (1986); Wetmur, Crit. Rev.Biochem. Mol. Biol., 26: 227-259 (1991);and the like. For shorter tags,e.g. about 30 nucleotides or less, the algorithm described by Rychlikand Wetmur is preferred, and for longer tags, e.g. about 30-35nucleotides or greater, an algorithm disclosed by Suggs et al, pages683-693 in Brown, editor, ICN-UCLA Symp. Dev. Biol., Vol. 23 (AcademicPress, New York, 1981) may be conveniently employed.

A preferred embodiment of minimally cross-hybridizing sets are thosewhose subunits are made up of three of the four natural nucleotides. Aswill be discussed more fully below, the absence of one type ofnucleotide in the oligonucleotide tags permits target polynucleotides tobe loaded onto solid phase supports by use of the 5'→3' exonucleaseactivity of a DNA polymerase. The following is an exemplary minimallycross-hybridizing set of subunits each comprising four nucleotidesselected from the group consisting of A, G, and T:

                  TABLE I    ______________________________________    Word       w.sub.1 w.sub.2   w.sub.3                                       w.sub.4    ______________________________________    Sequence:  GATT    TGAT      TAGA  TTTG    ______________________________________    Word:      w.sub.5 w.sub.6   w.sub.7                                       w.sub.8    ______________________________________    Sequence:  GTAA    AGTA      ATGT  AAAG    ______________________________________

In this set, each member would form a duplex having three mismatchedbases with the complement of every other member.

Further exemplary minimally cross-hybridizing sets are listed below inTable I. Clearly, additional sets can be generated by substitutingdifferent 10 groups of nucleotides, or by using subsets of knownminimally cross-hybridizing sets.

                  TABLE II    ______________________________________    Exemplary Minimally Cross-Hybridizing Sets of 4-mer Subunits    ______________________________________    CATT     ACCC     AAAC     AAAG   AACA   AACG    CTAA     AGGG     ACCA     ACCA   ACAC   ACAA    TCAT     CACG     AGGG     AGGC   AGGG   AGGC    ACTA     CCGA     CACG     CACC   CAAG   CAAC    TACA     CGAC     CCGC     CCGG   CCGC   CCGG    TTTC     GAGC     CGAA     CGAA   CGCA   CGCA    ATCT     GCAG     GAGA     GAGA   GAGA   GAGA    AAAC     GGCA     GCAG     GCAC   GCCG   GCCC             AAAA     GGCC     GGCG   GGAC   GGAG    AAGA     AAGC     AAGG     ACAG   ACCG   ACGA    ACAC     ACAA     ACAA     AACA   AAAA   AAAC    AGCG     AGCG     AGCC     AGGC   AGGC   AGCG    CAAG     CAAG     CAAC     CAAC   CACC   CACA    CCCA     CCCC     CCCG     CCGA   CCGA   CCAG    CGGC     CGGA     CGGA     CGCG   CGAG   CGGC    GACC     GACA     GACA     GAGG   GAGG   GAGG    GCGG     GCGG     GCGC     GCCC   GCAC   GCCC    GGAA     GGAC     GGAG     GGAA   GGCA   GGAA    ______________________________________

The oligonucleotide tags of the invention and their complements areconveniently synthesized on an automated DNA synthesizer, e.g. anApplied Biosystems, Inc. (Foster City, Calif.) model 392 or 394 DNA/RNASynthesizer, using standard chemistries, such as phosphoramiditechemistry, e.g. disclosed in the following references: Beaucage andIyer, Tetrahedron, 48: 2223-2311 (1992); MoLko et al, U.S. Pat. No.4,980,460; Koster et al, U.S. Pat. No. 4,725,677; Caruthers et al, U.S.Pat. Nos. 4,415,732; 4,458,066; and 4,973,679; and the like. Alternativechemistries, e.g. resulting in non-natural backbone groups, such asphosphorothioate, phosphoramidate, and the like, may also be employedprovided that the resulting oligonucleotides are capable of specifichybridization. In some embodiments, tags may comprise naturally occuringnucleotides that permit processing or manipulation by enzymes, while thecorresponding tag complements may comprise non-natural nucleotideanalogs, such as peptide nucleic acids, or like compounds, that promotethe formation of more stable duplexes during sorting.

When microparticles are used as supports, repertoires of oligonucleotidetags and tag complements are preferably generated by subunit-wisesynthesis via "split and mix" techniques, e.g. as disclosed in Shortleet al, International patent application PCTIUS93/03418. Briefly, thebasic unit of the synthesis is a subunit of the oligonucleotide tag.Preferably, phosphoramidite chemistry is used and 3' phosphoramiditeoligonucleotides are prepared for each subunit in a minimallycross-hybridizing set, e.g. for the set first listed above, there wouldbe eight 4-mer 3'-phosphoramidites. Synthesis proceeds as disclosed byShortle et al or in direct analogy with the techniques employed togenerate diverse oligonucleotide libraries using nucleosidic monomers,e.g. as disclosed in Telenius et al, Genomics, 13: 718-725 (1992); Welshet al, Nucleic Acids Research, 19: 5275-5279 (1991); Grothues et al,Nucleic Acids Research, 21: 1321-1322 (1993); Hartley, European patentapplication 90304496.4; Lam et al, Nature, 354: 82-84 (1991); Zuckermanet al, Int. J. Pept. Protein Research, 40: 498-507 (1992); and the like.Generally, these techniques simply call for the application of mixturesof the activated monomers to the growing oligonucleotide during thecoupling steps.

Double stranded forms of tags are made by separately synthesizing thecomplementary strands followed by mixing under conditions that permitduplex formation. Such duplex tags may then be inserted into cloningvectors along with target polynucleotides for sorting and manipulationof the target polynucleotide in accordance with the invention.

In embodiments where specific hybridization occurs via triplexformation, coding of tag sequences follows the same principles as forduplex-forming tags; however, there are further constraints on theselection of subunit sequences. Generally, third strand association viaHoogsteen type of binding is most stable along homopyrimidine-homopurinetracks in a double stranded target. Usually, base triplets form in T-A*Tor C-G*C motifs (where "-" indicates Watson-Crick pairing and "*"indicates Hoogsteen type of binding); however, other motifs are alsopossible. For example, Hoogsteen base pairing permits parallel andantiparallel orientations between the third strand (the Hoogsteenstrand) and the purine-rich strand of the duplex to which the thirdstrand binds, depending on conditions and the composition of thestrands. There is extensive guidance in the literature for selectingappropriate sequences, orientation, conditions, nucleoside type (e.g.whether ribose or deoxyribose nucleosides are employed), basemodifications (e.g. methylated cytosine, and the like) in order tomaximize, or otherwise regulate, triplex stability as desired inparticular embodiments, e.g. Roberts et al, Proc. Natl. Acad. Sci., 88:9397-9401 (1991); Roberts et al, Science, 258: 1463-1466 (1992);Distefano et al, Proc. Natl. Acad. Sci., 90: 1179-1183 (1993); Mergny etal, Biochemistry, 30: 9791-9798 (1991); Cheng et al, J. Am. Chem. Soc.,114: 4465-4474 (1992); Beal and Dervan, Nucleic Acids Research, 20:2773-2776 (1992); Beal and Dervan, J. Am. Chem. Soc., 114: 4976-4982(1992); Giovannangeli et al, Proc. Natl. Acad. Sci., 89: 8631-8635(1992); Moser and Dervan, Science, 238: 645-650 (1987); McShan et al, J.Biol. Chem., 267:5712-5721 (1992); Yoon et al, Proc. Natl. Acad. Sci.,89: 3840-3844 (1992); Blume et al, Nucleic Acids Research, 20: 1777-1784(1992); Thuong and Helene, Angew. Chem. Int. Ed. Engl. 32: 666-690(1993); and the like. Conditions for annealing single-stranded or duplextags to their single-stranded or duplex complements are well known, e.g.Ji et al, Anal. Chem. 65: 1323-1328 (1993).

When oligomeric subunits are employed, oligonucleotide tags of theinvention and their complements may range in length from 12 to 60nucleotides or basepairs; more preferably, they range in length from 18to 40 nucleotides or basepairs; and most preferably, they range inlength from 25 to 40 nucleotides or basepairs. When constructed fromantisense monomers, oligonucleotide tags and their complementspreferably range in length from 10 to 40 monomers; and more preferably,they range in length from 12 to 30 monomers.

Solid Phase Supports

Solid phase supports for use with the invention may have a wide varietyof forms, including microparticles, beads, and membranes, slides,plates, micromachined chips, and the like. Likewise, solid phasesupports of the invention may comprise a wide variety of compositions,including glass, plastic, silicon, alkanethiolate-derivatized gold,cellulose, low cross-linked and high cross-linked polystyrene, silicagel, polyamide, and the like. Preferably, either a population ofdiscrete particles are employed such that each has a uniform coating, orpopulation, of complementary sequences of the same tag (and no other),or a single or a few supports are employed with spacially discreteregions each containing a uniform coating, or population, ofcomplementary sequences to the same tag (and no other). In the latterembodiment, the area of the regions may vary according to particularapplications; usually, the regions range in area from several μm², e.g.3-5, to several hundred μm², e.g. 100-500. Preferably, such regions arespacially discrete so that signals generated by events, e.g. fluorescentemissions, at adjacent regions can be resolved by the detection systembeing employed. In some applications, it may be desirable to haveregions with uniform coatings of more than one tag complement, e.g. forsimultaneous sequence analysis, or for bringing separately taggedmolecules into close proximity.

Tag complements may be used with the solid phase support that they aresynthesized on, or they may be separately synthesized and attached to asolid phase support for use, e.g. as disclosed by Lund et al, NucleicAcids Research, 16: 10861-10880 (1988); Albretsen et al, Anal. Biochem.,189: 40-50 (1990); Wolf et al, Nucleic Acids Research, 15: 2911-2926(1987); or Ghosh et al, Nucleic Acids Research, 15: 5353-5372 (1987).Preferably, tag complements are synthesized on and used with the samesolid phase support, which may comprise a variety of forms and include avariety of linking moieties. Such supports may comprise microparticlesor arrays, or matrices, of regions where uniform populations of tagcomplements are synthesized. A wide variety of microparticle supportsmay be used with the invention, including microparticles made ofcontrolled pore glass (CPG), highly cross-linked polystyrene, acryliccopolymers, cellulose, nylon, dextran, latex, polyacrolein, and thelike, disclosed in the following exemplary references: Meth. Enzymol.,Section A, pages 11-147, vol. 44 (Academic Press, New York, 1976); U.S.Pat. Nos. 4,678,814; 4,413,070; and 4,046;720; and Pon, Chapter 19, inAgrawal, editor, Methods in Molecular Biology, Vol. 20, (Humana Press,Totowa, N.J., 1993). Microparticle supports further include commerciallyavailable nucleoside-derivatized CPG and polystyrene beads (e.g.available from Applied Biosystems, Foster City, Calif.); derivatizedmagnetic beads; polystyrene grafted with polyethylene glycol (e.g.,TentaGel™, Rapp Polymere, Tubingen Germany); and the like. Selection ofthe support characteristics, such as material porosity, size, shape, andthe like, and the type of linking moiety employed depends on theconditions under which the tags are used. For example, in applicationsinvolving successive processing with enzymes, supports and linkers thatminimize steric hinderance of the enzymes and that facilitate access tosubstrate are preferred. Exemplary ling moieties are disclosed in Pon etal, Biotechniques, 6:768-775 (1988); Webb, U.S. Pat. No. 4,659,774;Barany et al, International patent application PCT/US91/06103; Brown etal, J. Chem. Soc. Commun., 1989: 891-893; Damha et al, Nucleic AcidsResearch, 18: 3813-3821 (1990); Beattie et al, Clinical Chemistry, 39:719-722 (1993); Maskos and Southern, Nucleic Acids Research, 20:1679-1684 (1992); and the like.

As mentioned above, tag complements may also be synthesized on a single(or a few) solid phase support to form an array of regions uniformlycoated with tag complements. That is, within each region in such anarray the same tag complement is synthesized. Techniques forsynthesizing such arrays are disclosed in McGall et al, Internationalapplication PCT/US93/03767; Pease et al, Proc. Natl. Acad. Sci., 91:5022-5026 (1994); Southern and Maskos, International applicationPCT/GB89/01114; Maskos and Southern (cited above); Southern et al,Genomics, 13: 1008-1017 (1992); and Maskos and Southern, Nucleic AcidsResearch, 21: 4663-4669 (1993).

Preferably, the invention is implemented with microparticles or beadsuniformly coated with complements of the same tag sequence.Microparticle supports and methods of covalently or noncovalentlylinking oligonucleotides to their surfaces are well known, asexemplified by the following references: Beaucage and Iyer (citedabove); Gait, editor, Oligonucleotide Synthesis: A Practical Approach(IRL Press, Oxford, 1984); and the references cited above. Generally,the size and shape of a microparticle is not critical; however,microparticles in the size range of a few, e.g. 1-2, to several hundred,e.g. 200-1000 μm diameter are preferable, as they facilitate theconstruction and manipulation of large repertoires of oligonucleotidetags with minimal reagent and sample usage.

Preferably, commercially available controlled-pore glass (CPG) orpolystyrene supports are employed as solid phase supports in theinvention. Such supports come available with base-labile linkers andinitial nucleosides attached, e.g. Applied Biosystems (Foster City,Calif.). Preferably, microparticles having pore size between 500 and1000 angstroms are employed.

Synthesis of Oligonucleotide N3'→P5' Phosphoramidates

Tag complements comprising oligonucleotide N3'→P5' phosphoramidates maybe synthesized on a solid support using the step-by-step elongationprocedure outlined in FIG. 4. The synthetic cycle for addition of asingle aminonucleoside consists essentially of the following operations:detritylation (step a); phosphitylation of the 5' hydroxyl group togenerate a 5'-hydrogen phosphonate diester (steps b and c); andAtherton-Todd type coupling of a 5'-DMT-3'-aminonucleoside (e.g. asdisclosed by Glinski et al, Chem. Comm., pp. 915-916 (1970)) with the 5'hydrogen phosphonate in the presence of carbon tetrachloride (step d).

Coupling yields range between 94-96% per cycle. The resultingoligonucleotide phosphoramidate is cleaved and deprotected with ammoniaand thereafter purified by ion exchange high performance liquidchromatography. The following references provide guidance for carryingout the above synthesis: Atherton et al, J. Chem. Soc., pp. 660-663(1945); Gryaznov et al, Nucleic Acids Research, 20: 3403-3409 (1992);Gryaznov et al, Vest. Mosk. Univ. Ser. 2: Khim 27: 421-424 (1986); andGryaznov et al, Tetrahedron Lett., 31: 3205-3208 (1990). Fung andGryaznov, International application PCT/US94/03087, also show that3'-amino-oligonucleotides may be enzymatically ligated to5'-phosphorylated oligonucleotides in a standard template-drivenligation reaction.

Oligonucleotide Clamps

Tag complements of the invention may comprise an oligonucleotide clamp,which is a compound capable of forming a covalently closed macrocycle ora stable circular complex after specifically binding to a targetpolynucleotide, which in the case of the present invention is itscorresponding oligonucleotide tag. Generally, oligonucleotide clampscomprise one or more oligonucleotide moieties capable of specificallybinding to a tag and one or more pairs of binding moieties covalentlylinked to the oligonucleotide moieties. Upon annealing of theoligonucleotide moieties to the target polynucleotide, the bindingmoieties of a pair are brought into juxtaposition so that they form astable covalent or non-covalent linkage or complex. The interaction ofthe binding moieties of the one or more pairs effectively clamps thespecifically annealed oligonucleotide moieties to the targetpolynucleotide.

In one preferred form oligonucleotide clamps comprise a first bindingmoiety, a first oligonucleotide moiety, a hinge region, a secondoligonucleotide moiety, and a second binding moiety, for example, asrepresented by the particular embodiment of the following formula:

    X--O.sub.1 --G--O.sub.2 --Y

wherein O₁ and O₂ are the first and second oligonucleotide moieties, Gis the hinge region, X is the first binding moiety and Y is the secondbinding moiety such that X and Y form a stable covalent or non-covalentlinkage or complex whenever they are brought into juxtapositon by theannealing of the oligonucleotide moieties to a target polynucleotide.Preferably, in this embodiment, one of O₁ and O₂ undergoes Watson-Crickbinding with the target polynucleotide while the other of O₁ and O₂undergoes Hoogsteen binding.

Preferably, stability of oligonucleotide clamp/target polynucleotidecomplexes are determined by way of melting, or strand dissociation,curves. The temperature of fifty percent strand dissociation is taken asthe melting temperature, T_(m), which, in turn, provides a convenientmeasure of stability. T_(m) measurements are typically carried out in asaline solution at neutral pH with target and clamp concentrations atbetween about 1.0-2.0 μM. Typical conditions are as follows: 150 mM NaCland 10 mM MgCl₂ in a 10 mM sodium phosphate buffer (pH 7.0) or in a 10mM Tris-HCI buffer (pH 7.0); or like conditions. Data for melting curvesare accumulated by heating a sample of the oligonucleotide clamp/targetpolynucleotide complex from room temperature to about 85°-90° C. As thetemperature of the sample increases, absorbance of 260 nm light ismonitored at 1° C. intervals, e.g. using a Cary (Australia) model 1E ora Hewlett-Packard (Palo Alto, Calif.) model HP 8459 UV/VISspectrophotometer and model HP 89100A temperature controller, or likeinstruments.

Hinge regions consist of nucleosidic or non-nucleosidic polymers whichpreferably facilitate the specific binding of the monomers of theoligonucleotide moieties with their complementary nucleotides of thetarget polynucleotide. Hinge regions may also include Linkages to solidphase supports, e.g. via a derivatized base of a nucleotide, or thelike. Generally, the oligonucleotide moieties may be connected to hingeregions and/or binding moieties in either 5'→3' or 3'→5' orientations.For example, in the embodiment described above comprising a firstbinding moiety, a first oligonucleotide moiety, a hinge region, a secondoligonucleotide moiety, and a second binding moiety, the oligonucleotidemoieties may have any of the following orientations:

    X-(5')N.sub.1 N.sub.2 N.sub.3 - . . . -N.sub.j (3')-G-(5')N.sub.1 N.sub.2 N.sub.3 - . . . -N.sub.k (3')-Y

OR

    X-(5')N.sub.1 N.sub.2 N.sub.3 - . . . -N.sub.j (3')-G-(3')N.sub.k N.sub.k-1 N.sub.k-2 - . . . -N.sub.1 (5')-Y

OR

    X-(3')N.sub.j N.sub.j-1 N.sub.j-2 - . . . -N.sub.1 (5')-G-(5')N.sub.1 N.sub.2 N.sub.3 - . . . -N.sub.k (3')-Y

OR

    X-(3')N.sub.j N.sub.j-1 N.sub.j-2 - . . . -N.sub.1 (5')-G-(3')N.sub.k N.sub.k-1 N.sub.k-2 - . . . -N.sub.1 (5')-Y

wherein N₁ N₂ N₃ - . . . -N_(k) and N₁ N₂ N₃ - . . . -N_(j) are k-merand j-mer oligonucleotide moieties in the indicated orientations.

Preferably, the hinge region is a linear oligomer of monomers selectedfrom the group consisting of alkyl, alkenyl, and/or ethers containing2-3 carbon atoms.

Preferably, for nucleoside-sized monomers or smaller, the number ofmonomers varies between about 3 and about 10; and more preferably, itvaries between about 4 and 8.

A variety of binding moieties are suitable for use with the invention.Generally, they are employed in pairs, which for convenience here willbe referred to as X and Y. X and Y may be the same or different.Whenever the interaction of X and Y is based on the formation of stablehydrophobic complex, X and Y are lipophilic groups, including alkylgroups, fatty acids, fatty alcohols, steroids, waxes, fat-solublevitamins, and the like. Further exemplary lipophilic binding moietiesinclude glycerides, glyceryl ethers, phospholipids, sphingolipids,terpenes, and the like. In such embodiments, X and Y are preferablyselected from the group of steroids consisting of a derivatizedperhydrocyclopentanophenanthrene nucleus having from 19 to 30 carbonatoms, and 0 to 6 oxygen atoms; alkyl having from 6 to 16 carbon atoms;vitamin E; and glyceride having 20 to 40 carbon atoms. Preferably, aperhydrocyclopentanophenanthrene-based moiety is attached through thehydroxyl group, either as an ether or an ester, at its C3 position. Itis understood tha X and Y may include a linkage group connecting it toan oligonucleotide moiety. For example, glyceride includesphosphoglyceride, e.g. as described by MacKellar et al, Nucleic AcidsResearch, 20: 3411-3417 (1992), and so on. It is especially preferredthat lipophilic moieties, such as perhydrocyclopentanophenanthrenederivatives, be linked to the 5' carbon and/or the 3' carbon of anoligonucleotide moiety by a short but flexible linker that permits thelipophilic moiety to interact with the bases of the oligonucleotideclamp/target polynucleotide complex or a lipophilic moiety on the sameor another oligonucleotide moiety. Such linkers include phosphate (i.e.phosphodiester), phosphoramidate, hydroxyurethane, carboxyaminoalkyl andcarboxyaminoalkylphosphate linkers, or the like. Preferably, suchlinkers have no more than from 2 to 8 carbon atoms.

Binding moieties can be attached to the oligonucleotide moiety by anumber of available chemistries. Generally, it is preferred that theoligonucleotide be initially derivatized at its 3' and/or 5' terminuswith a reactive functionality, such as an amino, phosphate,thiophosphate, or thiol group. After derivatization, a hydrophilic orhydrophobic moiety is coupled to the oligonucleotide via the reactivefunctionality. Exemplary means for attaching 3' or 5' reactivefunctionalities to oligonucleotides are disclosed in Fung et al, U.S.Pat. No. 5,212,304; Connolly, Nucleic Acids Research, 13: 4485-4502(1985); Tino, International application PCT/US91/09657; Nelson et al,Nucleic Acids Research, 17: 7187-7194 (1989); Stabinsky, U.S. Pat. No.4,739,044; Gupta et al, Nucleic Acids Research, 19: 3019 (1991); Reed etal, International application PCT/US91/06143; Zuckerman et al, NucleicAcids Research, 15: 5305 (1987); Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991); Clontech1992/1993 Catalog (Clontech Laboratories, Palo Alto, Calif.); and likereferences.

Preferably, whenever X and Y form a covalent linkage, X and Y pairs mustreact specifically with each other when brought into juxtaposition. Inthis aspect of the invention, X and Y pairs are preferably selected fromthe following group: when one of X or Y is phosphorothioate orphosphorodithioate, the other is haloacetyl, haloacyl, haloalkyl, oralkylazide; when one of X or Y is thiol, the other is alkyl iodide,haloacyl, or haloacetyl; when one of Y or Y is phenylazide the other isphenylazide. More preferably, when one of X or Y is phosphorothioate orphosphorodithioate, the other is haloacetyl, haloacyl or haloalkyl,wherein said alkyl, acetyl, or acyl moiety contains from one to eightcarbon atoms. Such chemistries are disclosed by Gryaznov et al, J. Am.Chem. Soc., 115: 3808-3809 (1993).

In some embodiments, X and Y may form a covalent linkage in the presenceof an activating agent. That is, one or both of the binding moieties areactivated or rendered reactive towards one another by exposure to anactivating agent or condensing agent, such as radiation, a reducingagent, an oxidizing agent, or the like. Exemplary, binding moietiesemploying activating agents include thiophosphoryl groups in thepresence of K₃ Fe(CN)₆ or KI₃, e.g. Gryaznov and Letsinger, NucleicAcids Research, 21: 1403-1408 (1993); phosphoryl and hydroxyl in thepresence of N-cyanoimidazole, e.g. Luebke et al, J. Am. Chem. Soc., 113:7447-7448 (1991); phosphoryl or amino group and hydroxyl in the presenceof cyanogen bromide, e.g. Sokolova et al, FEBS Letters, 232: 153-155(1988); phosphoryl and hydroxyl groups in the presence ofspermine-5-(N-ethylimidazole)carboxamide and cyanoirnidazole, e.g. Zuberet al, J. Am. Chem. Soc., 115: 4939-4940 (1993); and the like.

Attaching Target Polynucleotides to Microparticles

An important aspect of the invention is the sorting of populations ofidentical polynucleotides, e.g. from a cDNA library, and theirattachment to microparticles or separate regions of a solid phasesupport such that each microparticle or region has only a single kind ofpolynucleotide. This latter condition can be essentially met by ligatinga repertoire of tags to a population of polynucleotides followed bycloning and sampling of the ligated sequences. A repertoire ofoligonucleotide tags can be ligated to a population of polynucleotidesin a number of ways, such as through direct enzymatic ligation,amplification, e.g. via PCR, using primers containing the tag sequences,and the like. The initial ligating step produces a very large populationof tag-polynucleotide conjugates such that a single tag is generallyattached to many different polynucleotides. However, by taking asufficiently small sample of the conjugates, the probability ofobtaining "doubles," i.e. the same tag on two different polynucleotides,can be made negligible. (Note that it is also possible to obtaindifferent tags with the same polynucleotide in a sample. This case issimply leads to a polynucleotide being processed, e.g. sequenced,twice). As explain more filly below, the probability of obtaining adouble in a sample can be estimated by a Poisson distribution since thenumber of conjugates in a sample will be large, e.g. on the order ofthousands or more, and the probability of selecting a particular tagwill be small because the tag repertoire is large, e.g. on the order oftens of thousand or more. Generally, the larger the sample the greaterthe probability of obtaining a double. Thus, a design trade-off existsbetween selecting a large sample of tag-polynucleotideconjugates--which, for example, ensures adequate coverage of a targetpolynucleotide in a shotgun sequencing operation, and selecting a smallsample which ensures that a minimal number of doubles will be present.In most embodiments, the presence of doubles merely adds an additionalsource of noise or, in the case of sequencing, a minor complication inscanning and signal processing, as microparticles giving multiplefluorescent signals can simply be ignored. As used herein, the term"substantially all" in reference to attaching tags to molecules,especially polynucleotides, is meant to reflect the statistical natureof the sampling procedure employed to obtain a population oftag-molecule conjugates essentially free of doubles. The meaning ofsubstantially all in terms of actual percentages of tag-moleculeconjugates depends on how the tags are being employed. Preferably, fornucleic acid sequencing, substantially all means that at least eightypercent of the tags have unique polynucleotides attached. Morepreferably, it means that at least ninety percent of the tags haveunique polynucleotides attached. Still more preferably, it means that atleast ninety-five percent of the tags have unique polynucleotidesattached. And, most preferably, it means that at least ninety-ninepercent of the tags have unique polynucleotides attached.

Preferably, when the population of polynucleotides is messenger RNA(mRNA), oligonucleotides tags are attached by reverse transcribing themRNA with a set of primers containing complements of tag sequences. Anexemplary set of such primers could have the following sequence (SEQ IDNO: 1):

    5'-mRNA-  A!.sub.n -3' T!.sub.19 GG W,W,W,C!.sub.9 ACCAGCTGATC-5'-biotin

where " W,W,W,C!₉ " represents the sequence of an oligonucleotide tag ofnine subunits of four nucleotides each and " W,W,W,C!" represents thesubunit sequences listed above, i.e. "W" represents T or A. Theunderlined sequences identify an optional restriction endonuclease sitethat can be used to release the polynucleotide from attachment to asolid phase support via the biotin, if one is employed. For the aboveprimer, the complement attached to a microparticle could have the form:

    5'- G,W,W,W!.sub.9 TGG-linker-microparticle

After reverse transcription, the mRNA is removed, e.g. by RNase Hdigestion, and the second strand of the cDNA is synthesized using, forexample, a primer of the following form (SEQ ID NO: 2):

    5'-NRRGATCYNNN-3'

where N is any one of A, T, G, or C; R is a purine-containingnucleotide, and Y is a pyrimidine-containing nucleotide. This particularprimer creates a Bst Y1 restriction site in the resulting doublestranded DNA which, together with the Sal I site, facilitates cloninginto a vector with, for example, Bam HI and Xho I sites. After Bst Y1and Sal I digestion, the exemplary conjugate would have the form (SEQ IDNO: 23):

    5'-RCGACCA C,W,W,W!.sub.9 GG T!.sub.19 -cDNA-NNNR GGT G,W,W,W!.sub.9 CC A!.sub.19 -rDNA-NNNYCTAG-5'

Preferably, when the ligase-based method of sequencing is employed, theBst Y1and Sal I digested fragments are cloned into a Bam HI-/XhoI-digested vector having the following single-copy restriction sites(SEQ ID NO: 3): ##STR1## This adds the Fok I site which will allowinitiation of the sequencing process discussed more fully below.

A general method for exposing the single stranded tag afteramplification involves digesting a target polynucleotide-containingconjugate with the 5'→3' exonuclease activity of T4 DNA polymerase, or alike enzyme. When used in the presence of a single nucleosidetriphosphate, such a polymerase will cleave nucleotides from 3' recessedends present on the non-template strand of a double stranded fragmentuntil a complement of the single nucleoside triphosphate is reached onthe template strand. When such a nucleotide is reached the 5'→3'digestion effectively ceases, as the polymerase's extension activityadds nucleotides at a higher rate than the excision activity removesnucleotides. Consequently, tags constructed with three nucleotides arereadily prepared for loading onto solid phase supports.

The technique may also be used to preferentially methylate interior FokI sites of a target polynucleotide while leaving a single Fok I site atthe terminus of the polynucleotide unmethylated. First, the terminal FokI site is rendered single stranded using a polymerase with deoxycytidinetriphosphate. The double standed portion of the fragment is thenmethylated, after which the single stranded terminus is filled in with aDNA polymerase in the presence of all four nucleoside triphosphates,thereby regenerating the Fok I site.

After the oligonucleotide tags are prepared for specific hybridization,e.g. by rendering them single stranded as described above, thepolynucleotides are mixed with microparticles containing thecomplementary sequences of the tags under conditions that favor theformation of perfectly matched duplexes between the tags and theircomplements. There is extensive guidance in the literature for creatingthese conditions. Exemplary references providing such guidance includeWetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991); Sambrook et al, Molecular Cloning: A Laboratory Manual,2nd Edition (Cold Spring Harbor Laboratory, New York, 1989); and thelike. Preferably, the hybridization conditions are sufficientlystringent so that only perfectly matched sequences form stable duplexes.Under such conditions the polynucleotides specifically hybridizedthrough their tags are ligated to the complementary sequences attachedto the microparticles. Finally, the microparticles are washed to removeunligated polynucleotides.

When CPG microparticles conventionally employed as synthesis supportsare used, the density of tag complements on the microparticle surface istypically greater than that necessary for some sequencing operations.That is, in sequencing approaches that require successive treatment ofthe attached polynucleotides with a variety of enzymes, densely spacedpolynucleotides may tend to inhibit access of the relatively bulkyenzymes to the polynucleotides. In such cases, the polynucleotides arepreferably mixed with the microparticles so that tag complements arepresent in significant excess, e.g. from 10:1 to 100:1, or greater, overthe polynucleotides. This ensures that the density of polynucleotides onthe microparticle surface will not be so high as to inhibit enzymeaccess. Preferably, the average inter-polynucleotide spacing on themicroparticle surface is on the order of 30-100 nm. Guidance inselecting ratios for standard CPG supports and Ballotini beads (a typeof solid glass support) is found in Maskos and Southern, Nucleic AcidsResearch, 20: 1679-1684 (1992). Preferably, for sequencing applications,standard CPG beads of diameter in the range of 20-50 μm are loaded withabout 10⁵ polynucleotides.

The above method may be used to fingerprint mRNA populations whencoupled with the parallel sequencing methodology described below.Partial sequence information is obtained simultaneously from a largesample, e.g. ten to a hundred thousand, of cDNAs attached to separatemicroparticles as described in the above method. The frequencydistribution of partial sequences can identify mRNA populations fromdifferent cell or tissue types, as well as from diseased tissues, suchas cancers. Such mRNA fingerprints are useful in monitoring anddiagnosing disease states.

Single Base DNA Sequencing

The present invention can be employed with conventional methods of DNAsequencing, e.g. as disclosed by Hultman et al, Nucleic Acids Research,17: 4937-4946 (1989). However, for parallel, or simultaneous, sequencingof multiple polynucleotides, a DNA sequencing methodology is preferredthat requires neither electrophoretic separation of closely sized DNAfragments nor analysis of cleaved nucleotides by a separate analyticalprocedure, as in peptide sequencing. Preferably, the methodology permitsthe stepwise identification of nucleotides, usually one at a time, in asequence through successive cycles of treatment and detection. Suchmethodologies are referred to herein as "single base" sequencingmethods. Single base approaches are disclosed in the followingreferences: Cheeseman, U.S. Pat. No. 5,302,509; Tsien et al,International application WO 91/06678; Rosenthal et al, Internationalapplication WO 93/21340; Canard et al, Gene, 148: 1-6 (1994); andMetzker et al, Nucleic Acids Research, 22: 4259-4267 (1994).

A "single base" method of DNA sequencing which is suitable for use withthe present invention and whch requires no electrophoretic separation ofDNA fragments is described in co-pending U.S. patent application Ser.No. 08/280,441 filed Jul. 25, 1994, which application is incorporated byreference. The method comprises the following steps: (a) ligating aprobe to an end of the polynucleotide having a protruding strand to forma ligated complex, the probe having a complementary protruding strand tothat of the polynucleotide and the probe having a nuclease recognitionsite; (b) removing unligated probe from the ligated complex; (c)identifying one or more nucleotides in the protruding strand of thepolynucleotide by the identity of the ligated probe; (d) cleaving theligated complex with a nuclease; and (e) repeating steps (a) through (d)until the nucleotide sequence of the polynucleotide is determined. As isdescribed more fully below, identifying the one or more nucleotides canbe carried out either before or after cleavage of the ligated complexfrom the target polynucleotide. Preferably, whenever natural proteinendonucleases are employed, the method further includes a step ofmethylating the target polynucleotide at the start of a sequencingoperation.

An important feature of the method is the probe ligated to the targetpolynucleotide. A preferred form of the probes is illustrated in FIG. 2.Generally, the probes are double stranded DNA with a protruding strandat one end 10. The probes contain at least one nuclease recognition site12 and a spacer region 14 between the recognition site and theprotruding end 10. Preferably, probes also include a label 16, which inthis particular embodiment is illustrated at the end opposite of theprotruding strand The probes may be labeled by a variety of means and ata variety of locations, the only restriction being that the labelingmeans selected does not interfer with the ligation step or with therecognition of the probe by the nuclease.

It is not critical whether protruding strand 10 of the probe is a 5' or3' end. However, it is important that the protruding strands of thetarget polynucleotide and probes be capable of forming perfectly matchedduplexes to allow for specific ligation. If the protruding strands ofthe target polynucleotide and probe are different lengths the resultinggap can be filled in by a polymerase prior to ligation, e.g. as in "gapLCR" disclosed in Backman et al, European patent application 91100959.5.Preferably, the number of nucleotides in the respective protrudingstrands are the same so that both strands of the probe and targetpolynucleotide are capable of being ligated without a filling step.Preferably, the protruding strand of the probe is from 2 to 6nucleotides long. As indicated below, the greater the length of theprotruding strand, the greater the complexity of the probe mixture thatis applied to the target polynucleotide during each ligation andcleavage cycle.

The complementary strands of the probes are conveniently synthesized onan automated DNA synthesizer, e.g. an Applied Biosystems, Inc. (FosterCity, Calif.) model 392 or 394 DNAIRNA Synthesizer, using standardchemistries. After synthesis, the complementary strands are combined toform a double stranded probe. Generally, the protruding strand of aprobe is synthesized as a mixture, so that every possible sequence isrepresented in the protruding portion. For example, if the protrudingportion consisted of four nucleotides, in one embodiment four mixturesare prepared as follows:

    X.sub.1 X.sub.2 . . . X.sub.i NNNA,

    X.sub.1 X.sub.2 . . . X.sub.i NNNC,

    X.sub.1 X.sub.2 . . . X.sub.i NNNG, and

    X.sub.1 X.sub.2 . . . X.sub.i NNNT

where the "NNNs" represent every possible 3-mer and the "Xs" representthe duplex forming portion of the strand. Thus, each of the four probeslisted above contains 4³ or 64 distinct sequences; or, in other words,each of the four probes has a degeneracy of 64. For example, X₁ X₂. . .X_(i) NNNA contains the following sequences: ##STR2## Such mixtures arereadily synthesized using well known techniques, e.g. as disclosed inTelenius et al (cited above). Generally, these techniques simply callfor the application of mixtures of the activated monomers to the growingoligonucleotide during the coupling steps where one desires to introducethe degeneracy. In some embodiments it may be desirable to reduce thedegeneracy of the probes. This can be accomplished using degeneracyreducing analogs, such as deoxyinosine, 2-aminopurine, or the like, e.g.as taught in Kong Thoo Lin et al, Nucleic Acids Research, 20: 5149-5152,or by U.S. Pat. No. 5,002,867.

Preferably, for oligonucleotides with phosphodiester linkages, theduplex forming region of a probe is between about 12 to about 30basepairs in length; more preferably, its length is between about 15 toabout 25 basepairs.

When conventional ligases are employed in the invention, as describedmore fully below, the 5' end of the probe may be phosphorylated in someembodiments. A 5' monophosphate can be attached to a secondoligonucleotide either chemically or enzymatically with a kinase, e.g.Sambrook et al (cited above). Chemical phosphorylation is described byHorn and Urdea, Tetrahedron Lett., 27: 4705 (1986), and reagents forcarrying out the disclosed protocols are commercially available, e.g. 5'Phosphate-ON™ from Clontech Laboratories (Palo Alto, Calif.). Thus, insome embodiments, probes may have the form:

    5'-X.sub.1 X.sub.2 . . . X.sub.i TTGA Y.sub.1 Y.sub.2. . . Y.sub.i p

where the Y's are the complementary nucleotides of the X's and "p" is amonophosphate group.

The above probes can be labeled in a variety of ways, including thedirect or indirect attachment of radioactive moieties, fluorescentmoieties, colorimetric moieties, chemiluminescent markers, and the like.Many comprehensive reviews of methodologies for labeling DNA andconstructing DNA probes provide guidance applicable to constructingprobes of the present invention. Such reviews include Kricka, editor,Nonisotopic DNA Probe Techniques (Academic Press, San Diego, 1992);Haugland, Handbook of Fluorescent Probes and Research Chemicals(Molecular Probes, Inc., Eugene, 1992); Keller and Manak, DNA Probes,2nd Edition (Stockton Press, New York, 1993); and Eckstein, editor,Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford,1991); Kessler, editor, Nonradioactive Labeling and Detection ofBiomolecules (Springer-Verlag, Berlin, 1992); Wetmur (cited above); andthe like.

Preferably, the probes are labeled with one or more fluorescent dyes,e.g. as disclosed by Menchen et al, U.S. Pat. No. 5,188,934; Begot et alInternational application PCT/US90/05565.

In accordance with the method, a probe is ligated to an end of a targetpolynucleotide to form a ligated complex in each cycle of ligation andcleavage. The ligated complex is the double stranded structure formedafter the protruding strands of the target polynucleotide and probeanneal and at least one pair of the identically oriented strands of theprobe and target are ligated, i.e. are caused to be covalently linked toone another. Ligation can be accomplished either enzymatically orchemically. Chemical ligation methods are well known in the art, e.g.Ferris et al, Nucleosides & Nucleotides, 8: 407-414 (1989); Shabarova etal, Nucleic Acids Research, 19: 4247--4251 (1991); and the like.Preferably, however, ligation is carried out enzymatically using aligase in a standard protocol. Many ligases are known and are suitablefor use in the invention, e.g. Lehman, Science, 186: 790-797 (1974);Engler et al, DNA Ligases, pages 3-30 in Boyer, editor, The Enzymes,Vol. 15B (Academic Press, New York, 1982); and the like. Preferredligases include T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, Taqligase, Pfu ligase, and Tth ligase. Protocols for their use are wellknown, e.g. Sambrook et al (cited above); Barany, PCR Methods andApplications, 1: 5-16 (1991); Marsh et al, Strategies, 5: 73-76 (1992);and the like. Generally, ligases require that a 5' phosphate group bepresent for ligation to the 3' hydroxyl of an abutting strand. This isconveniently provided for at least one strand of the targetpolynucleotide by selecting a nuclease which leaves a 5' phosphate, e.g.as Fok I.

In an embodiment of the sequencing method employing unphosphorylatedprobes, the step of ligating includes (i) ligating the probe to thetarget polynucleotide with a ligase so that a ligated complex is formedhaving a nick on one strand, (ii) phosphorylating the 5' hydroxyl at thenick with a kinase using conventional protocols, e.g. Sambrook et al(cited above), and (iii) ligating again to covalently join the strandsat the nick, i.e. to remove the nick.

Apparatus for Observing Enzymatic Processes and/or Binding Events atMicroparticle Surfaces

An objective of the invention is to sort identical molecules,particularly polynucleotides, onto the surfaces of microparticles by thespecific hybridization of tags and their complements. Once such sortinghas taken place, the presence of the molecules or operations performedon them can be detected in a number of ways depending on the nature ofthe tagged molecule, whether microparticles are detected separately orin "batches," whether repeated measurements are desired, and the like.Typically, the sorted molecules are exposed to ligands for binding, e.g.in drug development, or are subjected chemical or enzymatic processes,e.g. in polynucleotide sequencing. In both of these uses it is oftendesirable to simultaneously observe signals corresponding to such eventsor processes on large numbers of microparticles. Microparticles carryingsorted molecules (referred to herein as "loaded" microparticles) lendthemselves to such large scale parallel operations, e.g. as demonstratedby Lam et al (cited above).

Preferably, whenever light-generating signals, e.g. chemiluminescent,fluorescent, or the like, are employed to detect events or processes,loaded microparticles are spread on a planar substrate, e.g. a glassslide, for examination with a scanning system, such as described inInternational patent applications PCT/US91/09217 and PCT/NL90/00081. Thescanning system should be able to reproducibly scan the substrate and todefine the positions of each microparticle in a predetermined region byway of a coordinate system. In polynucleotide sequencing applications,it is important that the positional identification of microparticles berepeatable in successive scan steps.

Such scanning systems may be constructed from commercially availablecomponents, e.g. x-y translation table controlled by a digital computerused with a detection system consisting of one or more photomultipliertubes and appropriate optics, e.g. for exciting, collecting, and sortingfluorescent signals. Such a scanning system suitable for use infour-color sequencing is illustrated diagrammatically in FIG. 5.Substrate 300, e.g. a microscope slide with fixed microparticles, isplaced on x-y translation table 302, which is connected to andcontrolled by an appropriately programmed digital computer 304 which maybe any of a variety of commercially available personal computers, e.g.486-based machines or PowerPC model 7100 or 8100 available form AppleComputer (Cupertino, Calif.). Computer software for table translationand data collection functions can be provided by commercially availablelaboratory software, such as Lab Windows, available from NationalInstruments.

Substrate 300 and table 302 are operationally associated with microscope306 having one or more objective lenses 308 which are capable ofcollecting and delivering light to microparticles fixed to substrate300. Excitation beam 310 from light source 312, which is preferably alaser, is directed to beam splitter 314, e.g. a dichroic mirror, whichre-directs the beam through microscope 306 and objective lens 308 which,in turn, focuses the beam onto substrate 300. Lens 308 collectsfluorescence 316 emitted from the microparticles and directs it throughbeam splitter 314 to signal distribution optics 318 which, in turn,directs fluorescence to one or more suitable opto-electronic devices forconverting some fluorescence characteristic, e.g. intensity, lifetime,or the like, to an electrical signal. Signal distribution optics 318 maycomprise a variety of components standard in the art, such as bandpassfilters, fiber optics, rotating mirrors, fixed position mirrors andlenses, diffraction gratings, and the like. As illustrated in FIG. 5,signal distribution optics 318 directs fluorescence 316 to four separatephotomultiplier tubes, 330, 332, 334, and 336, whose output is thendirected to pre-amps and photon counters 350, 352, 354, and 356. Theoutput of the photon counters is collected by computer 304, where it canbe stored, analyzed, and viewed on video 360. Alternatively, signaldistribution optics 318 could be a diffraction grating which directsfluorescent signal 318 onto a CCD array.

The stability and reproducibility of the positional localization inscanning will determine, to a large extent, the resolution forseparating closely spaced microparticles. Preferably, the scanningsystems should be capable of resolving closely spaced microparticles,e.g. separated by a particle diameter. Thus, for most applications, e.g.using CPG microparticles, the scanning system should at least have thecapability of resolving objects on the order of 10-100 μm. Even higherresolution may be desirable in some embodiments, but with increaseresolution, the time required to fully scan a substrate will increase;thus, in some embodiments a compromise may have to be made between speedand resolution. Increases in scanning time can be achieved by a systemwhich only scans positions where microparticles are known to be located,e.g from an initial full scan. Preferably, microparticle size andscanning system resolution are selected to permit resolution offluorescendy labeled microparticles randomly disposed on a plane at adensity between about ten thousand to one hundred thousandmicroparticles per cm².

In sequencing applications, loaded microparticles can be fixed to thesurface of a substrate in variety of ways. The fixation should be strongenough to allow the microparticles to undergo successive cycles ofreagent exposure and washing without significant loss. Preferably, whenthe substrate is glass, its surface is derivatized with an alkylaminolinker using commercially available reagents, e.g. Pierce Chemical,which in turn is cross-linked to avidin, again using conventionalchemistries, to form an avidinated surface. Biotin moieties can beintroduced to the loaded microparticles in a number of ways. Forexample, a fraction, e.g. 10-15 percent, of the cloning vectors used toattach tags to polynucleotides are engineered to contain a uniquerestriction site (providing sticky ends on digestion) immediatelyadjacent to the polynucleotide insert at an end of the polynucleotideopposite of the tag. The site is excised with the polynucleotide and tagfor loading onto microparticles. After loading, about 10-15 percent ofthe loaded polynucleotides will possess the unique restriction sitedistal from the microparticle surface. After digestion with theassociated restriction endonuclease, an appropriate double strandedadaptor containing a biotin moiety is ligated to the sticky end. Theresulting microparticles are then spread on the avidinated glass surfacewhere they become fixed via the biotin-avidin linkages.

Altematively and preferably when sequencing by ligation is employed, inthe initial ligation step a mixture of probes is applied to the loadedmicroparticle: a fraction of the probes contain a type IIs restrictionrecognition site, as required by the sequencing method, and a fractionof the probes have no such recognition site, but instead contain abiotin moiety at its non-ligating end. Preferably, the mixture comprisesabout 10-15 percent of the biotinylated probe.

Parallel Sequencing

The tagging system of the invention can be used with single basesequencing methods to sequence polynucleotides up to several kilobasesin length. The tagging system permits many thousands of fragments of atarget polynucleotide to be sorted onto one or more solid phase supportsand sequenced simultaneously. In accordance with a preferredimplementation of the method, a portion of each sorted fragment issequenced in a stepwise fashion on each of the many thousands of loadedmicroparticles which are fixed to a common substrate--such as amicroscope slide--associated with a scanning system, such as thatdescribed above. The size of the portion of the fragments sequenceddepends of several factors, such as the number of fragments generatedand sorted, the length of the target polynucleotide, the speed andaccuracy of the single base method employed, the number ofmicroparticles and/or discrete regions that may be monitoredsimultaneously; and the like. Preferably, from 12-50 bases areidentified at each microparticle or region; and more preferably, 18-30bases are identified at each microparticle or region. With thisinformation, the sequence of the target polynucleotide is determined bycollating the 12-50 base fragments via their overlapping regions, e.g.as described in U.S. Pat. No. 5,002,867. The following referencesprovide additional guidance in determining the portion of the fragmentsthat must be sequenced for successful reconstruction of a targetpolynucleotide of a given length: Drmanac et al, Genomics, 4: 114-128(1989); Bains, DNA Sequencing and Mapping, 4: 143-150 (1993); Bains,Genomics, 11: 294-301 (1991); Drmanac et al, J. Biomolecular Structureand Dynamics, 8: 1085-1102 (1991); and Pevzner, J. BiomolecularStructure and Dynamics, 7: 63-73 (1989). Preferably, the length of thetarget polynucleotide is between 1 and 50 kilobases. More preferably,the length is between 10 and 40 kilobases.

Fragments may be generated from a target polynucleotide in a variety ofways, including so-called "directed" approaches where one attempts togenerate sets of fragments covering the target polynucleotide withminimal overlap, and so-called "shotgun" approaches where randomlyoverlapping fragments are generated. Preferably, "shotgun" approaches tofragment generation are employed because of their simplicity andinherent redundancy. For example, randomly overlapping fragments thatcover a target polynucleotide are generated in the followingconventional "shotgun" sequencing protocol, e.g. as disclosed inSambrook et al (cited above). As used herein, "cover" in this contextmeans that every portion of the target polynucleotide sequence isrepresented in each size range, e.g. all fragments between 100 and 200basepairs in length, of the generated fragments. Briefly, starting witha target polynucleotide as an insert in an appropriate cloning vector,e.g. λ phage, the vector is expanded, purified and digested with theappropriate restriction enzymes to yield about 10-15 μg of purifedinsert. Typically, the protocol results in about 500-1000 subclones permicrogram of starting DNA. The insert is separated from the vectorfragments by preparative gel electrophoresis, removed from the gel byconventional methods, and resuspended in a standard buffer, such as TE(Tris-EDTA). The restriction enzymes selected to excise the insert fromthe vector preferably leave compatible sticky ends on the insert, sothat the insert can be self-ligated in preparation for generatingrandomly overlapping fragments. As explained in Sambrook et al (citedabove), the circularized DNA yields a better random distribution offragments than linear DNA in the fragmentation methods employed below.After self-ligating the insert, e.g. with T4 ligase using conventionalprotocols, the purifed ligated insert is fragmented by a standardprotocol, e.g. sonication or DNase I digestion in the presence of Mn⁺⁺.After fragmentation the ends of the fragments are repair, e.g. asdescribed in Sambrook et al (cited above), and the repaired fragmentsare separated by size using gel electrophoresis. Fragments in the300-500 basepair range are selected and eluted from the gel byconventional means, and ligated into a tag-carrying vector to form alibrary of tag-fragment conjugates.

As described above, a sample containing several thousand tag-fragmentconjugates are taken from the library and expanded, after which thetag-fragment inserts are excised from the vector and prepared forspecific hybridization to the tag complements on microparticles, asdescribed above. Depending of the size of the target polynucleotide,multiple samples may be taken from the tag-fragment library andseparately expanded, loaded onto microparticles and sequenced. Thenumber of doubles selected will depend on the fraction of the tagrepertoire represented in a sample. (The probability of obtainingtriples-three different polynucleotides with the same tag--or above cansafely be ignored). As mentioned above, the probability of doubles in asample can be estimated from the Poisson distribution p(double)=m²e^(-m) /2, where m is the fraction of the tag repertoire in the sample.Table IV below lists probabilities of obtaining doubles in a sample forgiven tag size, sample size, and repertoire diversity.

                  TABLE IV    ______________________________________    Number of                    Fraction of    words in tag              Size of tag                        Size     repertoire                                         Probability    from 8 word set              repertoire                        of sample                                 sampled of double    ______________________________________    7          2.1 × 10.sup.6                        3000     1.43 × 10.sup.-3                                         10.sup.-6    8         1.68 × 10.sup.7                        3 × 10.sup.4                                 1.78 × 10.sup.-3                                         1.6 × 10.sup.-6                        3000     1.78 × 10.sup.-4                                         1.6 × 10.sup.-8    9         1.34 × 108                        3 × 10.sup.5                                 2.24 × 10.sup.-3                                         2.5 × 10.sup.-6                        3 × 10.sup.4                                 2.24 × 10.sup.-4                                         2.5 × 10.sup.-8    10        1.07 × 10.sup.9                        3 × 10.sup.6                                  2.8 × 10.sup.-3                                         3.9 × 10.sup.-6                        3 × 10.sup.5                                  2.8 × 10.sup.-4                                         3.9 × 10.sup.-8    ______________________________________

In any case, the loaded microparticles are then dispersed and fixed ontoa glass microscope slide, preferably via an avidin-biotin coupling.Preferably, at least 15-20 nucleotides of each of the random fragmentsare simultaneously sequenced with a single base method. The sequence ofthe target polynucleotide is then reconstructed by collating the partialsequences of the random fragments by way of their overlapping portions,using algorithms similar to those used for assembling contigs, or asdeveloped for sequencing by hybridization, disclosed in the abovereferences.

EXAMPLE 1 Sorting Multiple Target Polynucleotides Derived from pUC19with Tags having Oligomeric Subunits

A mixture of three target polynucleotide-tag conjugates are obtained asfollows: First, the following six oligonucleotides are synthesized andcombined pairwise to form tag 1, tag 2, and tag 3: ##STR3## where "p"indicates a monophosphate, the w_(i) 's represent the subunits define inTable I, and the terms "(**)" represent their respective complements. ApUC19 is digested with Sal I and Hind III, the large fragment ispurified, and separately ligated with tags 1, 2, and 3, to form pUC19-1,pUC19-2, and pUC19-3, respectively. The three recombinants areseparately amplified and isolated, after which pUC19-1 is digested withHind III and Aat I, pUC19-2 is digested with Hind III and Ssp I, andpUC19-3 is digested with Hind III and Xmn I. The small fragments areisolated using conventional protocols to give three double strandedfragments about 250, 375, and 575 basepairs in length, respectively, andeach having a recessed 3' strand adjacent to the tag and a blunt or 3'protruding strand at the opposite end. Approximately 12 nmoles of eachfragment are mixed with 5 units T4 DNA polymerase in the manufacturer'srecommended reaction buffer containing 33 μM deoxycytosine triphosphate.The reaction mixture is allowed to incubate at 37° C. for 30 minutes,after which the reaction is stopped by placing on ice. The fragments arethen purified by conventional means.

CPG microparticles (37-74 mm particle size, 500 angstrom pore size,Pierce Chemical) are derivatized with the linker disclosed by Maskos andSouthern, Nucleic Acids Research, 20: 1679-1684 (1992). After separatinginto three aliquots, the complements of tags 1, 2, and 3 are synthesizedon the microparticles using a conventional automated DNA synthesizer,e.g. a model 392 DNA synthesizer (Applied Biosystems, Foster City,Calif.). Approximately 1 mg of each of the differently derivatizedmicroparticles are placed in separate vessels.

The T4 DNA polymerase-treated fragments excised from pUC19-1, -2, and -3are resuspended in 50 μL of the manufacturer's recommended buffer forTaq DNA ligase (New England Biolabs). The mixture is then equallydivided among the three vessels containing the 1 mg each of derivatizedCPG microparticles. 5 units of Taq DNA ligase is added to each vessel,after which they are incubated at 55° C. for 15 minutes. The reaction isstopped by placing on ice and the microparticles are washed severaltimes by repeated centrifugation and resuspension in TE. Finally, themicroparticles are resuspended in Nde I reaction buffer (New EnglandBiolabs) where the attached polynucleotides are digested. Afterseparation from the microparticles the polynucleotide fragments releasedby Nde I digestion are fluorescently labeled by incubating withSequenase DNA polymerase and fluorescein labeled thymidine triphosphate(Applied Biosystems, Foster City, Calif.). The fragments are thenseparately analyzed on a nondenaturing polyacrylamide gel using anApplied Biosystems model 373 DNA sequencer.

EXAMPLE 2 Sorting Multiple Target Polynucleotides Derived from pUC19with Tag Complements Consisting of Oligonucleotide N3'→P5'Phosphoramidates

A mixture of three target polynucleotide-tag conjugates are obtained asfollows: First, the following six oligonucleotides are synthesized andcombined pairwise to form tag 1, (SEQ ID NO: 4 and SEQ ID NO: 5) tag 2,(SEQ ID NO: 6 and SEQ ID NO: 7) and tag 3 (SEQ ID NO: 8 and SEQ ID NO:9): ##STR4## Three 20-mer oligonucleotide N3'→P5' phosphoramidate tagcomplements are seperately synthesized on CPG microparticles asdescribed above, the tag complements having the following sequences (in3'→5' orientation): CTAACTAAACTAACTATACA (SEQ ID NO: 10),CATTTCATCTAAACTCAT (SEQ ID NO: 11), and ACTAACTATACATACACATT (SEQ ID NO:12). The polynucleotide-tag conjugates are prepared and sorted asdescribed in Example 4.

EXAMPLE 3 Parallel Sequencing of SV40 Fragments

A repertoire of 36-mer tags consisting of nine 4-nucleotide subunitsselected from Table I is prepared by separately synthesizing tags andtag complements by a split and mix approach, as described above. Therepertoire is synthesized so as to permit ligation into a Sma I/Hind IIIdigested M13mp19. Thus, as in Example I, one set of oligonucleotidesbegins with the addition of A followed by nine rounds of split and mixsynthesis wherein the oligonucleotide is extended subunit-wise by3'-phosphoramidite derivatived 4-mers corresponding to the subunits ofTable I. The synthesis is then completed with thenucleotide-by-nucleotide addition of one half of the Sma I recognitionsite (GGG), two C's, and a 5'-monophosphate, e.g. via the Phosphate-ONreagent available from Clontech Laboratories (Palo Alto, Calif.). Theother set of oligonucleotides begins with the addition of three C's(portion of the Sma I recognition site) and two G's, followed by ninerounds of split and mix synthesis wherein the oligonucleotide isextended by 3'-phosphoramidite derivatized 4-mers corresponding to thecomplements of the subunits of Table I. Synthesis is completed by thenucleotide-by-nucleotide addition of the Hind m recognition site and a5'-monophosphate. After separation from the synthesis supports theoligonucleotides are mixed under conditions that permit formation of thefollowing duplexes:

    5'-pGGGCC(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)(w.sub.i)A CCCGG(**)(**)(**)(**)(**)(**)(**)(**)(**)TTCGAp-5'

The mixture of duplexes is then ligated into a Sma I/Hind III-digestedM13mp19. A repertoire of tag complements are synthesized on CPGmicroparticles as described above.

Next the following adaptor is prepared which contains a Fok I site andportions of Eco RI and Sma I sites (SEQ ID NO: 13 and SEQ ID NO: 14):##STR5## The adaptor is ligated into the Eco RI/Sma I digested M13described above.

Separately, SV40 DNA is fragmented by sonication following the protocolset forth in Sambrook et al (cited above). The resulting fragments arerepaired using standard protocols and separated by size. Fragments inthe range of 300-500 basepairs are selected and ligated into the Sma Idigested M13 described above to form a library of fragment-tagconjugates, which is then amplified. A sample containing severalthousand different fragment-tag conjugates is taken from the library,further amplified, and the fragment-tag inserts are excised by digestingwith Eco RI and Hind III. The excised fragment-tag conjugates aretreated with T4 DNA polymerase in the presence of deoxycytidinetriphosphate, as described in Example 1, to expose the oligonucleotidetags for specific hybridization to the CPG microparticles.

After hybridization and ligation, as described in Example I, the loadedmicroparticles are treated with Fok I to produce a 4-nucleotideprotruding strand of a predetermined sequence. A 10:1 mixture (probe 1:probe 2) of the following probes are ligated to the polynucleotides onmicroparticles. ##STR6## FAM represents a fluorescein dye attached tothe 5'-hydroxyl of the top strand of Probe 1 through an aminophosphatelinker available from Applied Biosystems (Aminolinker). The biotin mayalso be attached through an Aminolinker moiety and optionally may befurther extended via polyethylene oxide linkers, e.g. Jaschke et al(cited above).

The loaded microparticles are then deposited on the surface of anavidinated glass slide to which and from which reagents and washsolutions can be delivered and removed. The avidinated slide with theattached microparticles is examined with a scanning fluorescentmicroscope (e.g. Zeiss Axioskop equipped with a Newport Model PM500-Cmotion controller, a Spectra-Physics Model 2020 argon ion laserproducing a 488 nm excitation beam, and a 520 nm long-pass emissionfilter, or like apparatus). The excitation beam and fluorescentemissions are delivered and collected, respectively, through the sameobjective lens. The excitation beam and collected fluorescence areseparated by a dichroic mirror which directs the collected fluorescencethrough a series of bandpass filters and to photon-counting devicescorresponding to the fluorophors being monitored, e.g. comprisingHamamatsu model 9403-02 photomultipliers, a Stanford Research Systemsmodel SR445 amplifier and model SR430 multichannel scaler, and digitalcomputer, e.g. a 486-based computer. The computer generates a twodimensional map of the slide which registers the positions of themicroparticles.

After cleavage with Fok I to remove the initial probe, thepolynucleotides on the attached microparticles undergo 20 cycles ofprobe ligation, washing, detection, cleavage, and washing, in accordancewith the preferred single base sequencing methodology described below.Within each detection step, the scanning system records the fluorescentemission corresponding the base identified at each microparticle.Reactions and washes below are generally carried out with manufacturer's(New England Biolabs') recommended buffers for the enzymes employed,unless otherwise indicated. Standard buffers are also described inSambrook et al (cited above).

The following four sets of mixed probes are provided for addition to thetarget polynucleotides (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, and SEQ ID NO: 21):

    TAMRA- ATCGGATGACATCAAC TAGCCTACTGTAGTTGANNN

    FAM- ATCGGATGACATCAAC TAGCCTACTGTAGTTGCNNN

    ROX- ATCGGATGACATCAAC TAGCCTACTGTAGTTGGNNN

    JOE- ATCGGATGACATCAAC TAGCCTACTGTAGTTGTNNN

where TAMRA, FAM, ROX, and JOE are spectrally resolvable fluorescentlabels attached by way of Aminolinker II (all being available fromApplied Biosystems, Inc., Foster City, Calif.); the bold facednucleotides are the recognition site for Fok I endonuclease, and "N"represents any one of the four nucleotides, A, C, G, T.TAMRA(tetramethylrhodamine), FAM(fluorescein), ROX(rhodamine X), and JOE(2',7'-dimethoxy-4',5'-dichlorofluorescein) and their attachment tooligonucleotides is also described in Fung et al, U.S. Pat. No.4,855,225.

The above probes are incubated in approximately 5 molar excess of thetarget polynucleotide ends as follows: the probes are incubated for 60minutes at 16° C. with 200 units of T4 DNA ligase and the anchoredtarget polynucleotide in T4 DNA ligase buffer; after washing, the targetpolynucleotide is then incubated with 100 units T4 polynucleotide kinasein the manufacturer's recommended buffer for 30 minutes at 37° C.,washed, and again incubated for 30 minutes at 16° C. with 200 units ofT4 DNA ligase and the anchored target polynucleotide in T4 DNA ligasebuffer. Washing is accomplished by successively flowing volumes of washbuffer over the slide, e.g. TE, disclosed in Sambrook et al (citedabove). After the cycle of ligation-phosphorylation-ligation and a finalwashing, the attached microparticles are scanned for the presence offluorescent label, the positions and characteristics of which arerecorded by the scanning system. The labeled target polynucleotide, i.e.the ligated complex, is then incubated with 10 units of Fok I in themanufacturer's recommended buffer for 30 minutes at 37° C., followed bywashing in TE. As a result the target polynucleotide is shortened by onenucleotide on each strand and is ready for the next cycle of ligationand cleavage. The process is continued until twenty nucleotides areidentified.

                  APPENDIX I    ______________________________________    Exemplary computer program for generating    minimally cross hybridizing sets    ______________________________________    Program minxh    c    c    integer*2 sub1(6),.mset1(1000,6),mset2(1000,6)    dimension nbase(6)    c    c            write(*,*) `ENTER SUBUNIT LENGTH`            read(*,100)nsub    100     format(i1)            Open(1,file=`sub4 dat`, form=`formatted`,status=`new`)    c    c            nset=0            do 7000.m1=1,3              do 7000 m2=1,3                do 7000 m3=1,3                  do 7000 m4=1,3                    sub1(1)=m1                    sub1(2)=m2                    sub1(3)=m3                    sub1(4)=m4    c    c            ndiff=3    c    c    c             Generate set of subunits differing from    c             sub1 by at least ndiff nucleotides.    c             Save in mset1.    c    c             jj=i             do 900 j=1,nsub    900        mseti(1,j)=sub1(j)    c    c             do 1000 k1=1,3               do 1000 k2=1,3                 do 1000 k3=1,3                   do 1000 k4=1,3    c    c                   nbase(1)=k1                   nbase(2)=k2                   nbase(3)=k3                  nbase(4) =k4    c                n=0                do 1200 j=1,nsub                    if(sub1(j).eq.1 .and. nbase(j).ne.1 .or.    1               sub1(j).eq.2 .and. nbase(j).ne.2 .or.    3               sub1(j).eq.3 .and. nbase(j).ne.3) then                   n=n+1                   endif    1200            continue    c    c            if(n.ge.ndiff) then    c    c    c                   If number of mismatches    c                   is greater than or equal    c                   to ndiff then record    c                   subunit in matrix mset    c    c                jj=jj+1                 do 1100 i=1,nsub    1100          #set1(jj,i)=nbase(i)                endif    c    c    1000    continue    c    c                do 1325 j2=1,nsub                mset2(1,j2)=mset1(1,j2)    1325        set2(2,j2)=mset1(2,j2).    c    c                   Compare subunit 2 from    c                   mseti with each successive    c                   subunit in mset1, i.e. 3,    c                   4,5, . . . etc. Save those    c                   with mismatches .ge. ndiff    c                   in matrix mset2 starting at    c                   position 2.    c                   Next transfer contents    c                   of mset2 into mset1 and    c                   start    c                   comparisons again this time    c                   starting with subunit 3.    c                   Continue until all subunits    c                   undergo the comparisons.    c    c            npass=0    c    c    1700    continue            kk=npass+2            npass=npass+1    c    c            do 1500 in=npass+2,jj              n=0              do 1600 j=1,nsub                if(mset1(npass+1,j) .eq.1.and.mset1(m,j) .ne. 1.or.    2           mset1(npass+1,j).eq.2.and.mset1(m,j).ne.2.or.    2           mset1(npass+1,j).eq.3.and.mset1(m,j).ne.3) then                n=n+1                endif    1600        continue                if (n.ge.ndiff) then                 kk=kk+1                 do 1625 i=1,nsub    1625           mset2(kk,i)=mset1(m,i)                endif    1500        continue    c    c    c                   kk is the number if subunits    c                   stored in mset2    c    c    c                   Transfer contents of mset2    c                   into mset1 for next pass.    c    c              do 2000 k=1,kk                do 2000 m=1,nsub    2000        mset1(k,m)=mset2(k,m)            if(kk.1t.jj) then             jj=kk             goto 1700             endif    c    c             nset=nset+1            write(1,7009)    7009     format(/)            do 7008 k=1,kk    7008        write(1,7010) (mset1(k,m),m=1,nsub)    7010    format(4i1)            write(*,*)            write(*,120) kk,nset    120     format(1x,`Subunits in set=`,i5,2x,`Set No=`,i5)    7000     continue             close(1)    c    c            end    ______________________________________

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 23    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    CTAGTCGACCA11    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    NRRGATCYNNN11    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 38 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    GAGGATGCCTTTATGGATCCACTCGAGATCCCAATCCA38    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    TCGACCGATTGATTTGATTGATATGTA27    (2) INFORMATION FOR SEQ ID NO: 5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    AGCTTACATATCAATCAAATCAATCG26    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    TCGACCGTAAAAAGTAGATTTGAGTAA27    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    AGCTTACTCAAATCTACTTTTTACGG26    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    TCGACCTGATTGATATGTATGTGTAAA27    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    AGCTTTACACATACATATCAATCAGG26    (2) INFORMATION FOR SEQ ID NO: 10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:    ACATATCAATCAAATCAATC20    (2) INFORMATION FOR SEQ ID NO: 11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:    TACTCAAATCTACTTTTTCA20    (2) INFORMATION FOR SEQ ID NO: 12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:    TTACACATACATATCAATCA20    (2) INFORMATION FOR SEQ ID NO: 13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 26 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:    AATTCGGATGATGCATGCATCGACCC26    (2) INFORMATION FOR SEQ ID NO: 14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 22 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:    GGGTCGATGCATGCATCATCCG22    (2) INFORMATION FOR SEQ ID NO: 15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:    ATCGGATGAC10    (2) INFORMATION FOR SEQ ID NO: 16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:    TCGAGTCATCCGAT14    (2) INFORMATION FOR SEQ ID NO: 17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:    ATCGGATGACATCAAC16    (2) INFORMATION FOR SEQ ID NO: 18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:    NNNAGTTGATGTCATCCGAT20    (2) INFORMATION FOR SEQ ID NO: 19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:    NNNCGTTGATGTCATCCGAT20    (2) INFORMATION FOR SEQ ID NO: 20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:    NNNGGTTGATGTCATCCGAT20    (2) INFORMATION FOR SEQ ID NO: 21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:    NNNTGTTGATGTCATCCGAT20    (2) INFORMATION FOR SEQ ID NO: 22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 37 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:    NNNNNGGATGNNNNNNNNNNNNNTNNNNNNNNNNNNN37    (2) INFORMATION FOR SEQ ID NO: 23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 21 nucleotides    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:    GGTTTTTTTTTTTTTTTTTTT21    __________________________________________________________________________

I claim:
 1. A method of characterizing a population of mRNA molecules,the method comprising the steps of:forming a population of cDNAmolecules from the population of mRNA molecules, the cDNA moleculesbeing complementary to the mRNA molecules and each cDNA molecule havingan oligonucleotide tag attached, (i) such that substantially alldifferent cDNA molecules have different oligonucleotide tags attachedand (ii) such that each oligonucleotide tag comprises a plurality ofsubunits and each subunit of the plurality consists of anoligonucleotide having a length from three to six nucleotides or fromthree to six basepairs, the subunits being selected from a minimallycross-hybridizing set wherein a subunit of the set and a complement ofany other subunit of the set would have at least two mismatches, sortingthe cDNA molecules by specifically hybridizing the oligonucleotide tagswith their respective complements, the respective complements beingattached as uniform populations of substantially identical complementsin spatially discrete regions on one or more solid phase supports andthe respective complements being oligodeoxyribonucleotide N3'→P5'phosphoramidates or peptide nucleic acids; determining the nucleotidesequence of a portion of each of the sorted cDNA molecules; andcharacterizing the population of mRNA molecules by the frequencydistribution of the portions of sequences of the cDNA molecules.
 2. Themethod of claim 1 wherein said one or more solid phase supports are aplurality of microparticles.
 3. The method of claim 2 wherein each saidportion of each of said sorted cDNA molecules is in the range of from 12to 50 nucleotides.
 4. The method of claim 3 wherein each said portion ofeach of said sorted cDNA molecules is in the range of from 12 to 25nucleotides.
 5. The method of claim 1 wherein said one or more solidphase supports are a plurality of microparticles.
 6. The method of claim5 wherein after said step of sorting, said plurality of microparticlesis fixed to a planar substrate.
 7. The method of claim 6 wherein saidplurality of microparticles are disposed randomly on the surface of saidplanar substrate at a density of between 1000 microparticles to 100thousand microparticles per square centimeter.
 8. The method of claim 1wherein each of said oligonucleotide tags has a length the range of from10 to 40 monomers.
 9. The method of claim 1 wherein said population ofcDNA molecules contains from 500 to 1000 cDNA molecules.
 10. A method ofcharacterizing a population of mRNA molecules, the method comprising thesteps of:forming a population of cDNA molecules from the population ofmRNA molecules, the cDNA molecules being complementary to the mRNAmolecules; attaching an oligonucleotide tag from a repertoire of tags toeach cDNA molecule of the population such that substantially alldifferent cDNA molecules have different oligonucleotide tags attached;sorting the cDNA molecules by specifically hybridizing theoligonucleotide tags with their respective complements, the respectivecomplements being attached as uniform populations of substantiallyidentical complements in spatially discrete regions on one or more solidphase supports and the respective complements beingoligodeoxyribonucleotide N3'→P5' phosphoramidates or peptide nucleicacids; determining the nucleotide sequence of a portion of each of thecDNA molecules of the population; and characterizing the population ofmRNA molecules by the frequency distribution of the portions ofsequences of the cDNA molecules.
 11. The method of claim 10 wherein saidoligonucleotide tags are single stranded oligonucleotides.
 12. Themethod of claim 11 wherein said step of determining said nucleotidesequence of said cDNA molecules is carried out simultaneously for saidpopulation of cDNA molecules by a single base sequencing method.
 13. Themethod of claim 12 wherein each said portion of each of said sorted cDNAmolecules is from 12 to 50 nucleotides in length.
 14. The method ofclaim 13 wherein each said portion of each of said sorted cDNA moleculesis from 12 to 25 nucleotides in length.